PROJECT SUMMARY The Hemagglutination assay (HA) and Hemagglutination Inhibition (HI) assay are widely used methods for characterizing viruses, bacteria, and antibodies. The World Health Organization recently recommended that HI remain the gold standard method for influenza seroepidemiology. Although the HA and HI assays are applied worldwide, they have the disadvantages of manual plate reading, non- specific inhibition, and other operational limitations causing poor reproducibility and inconsistent results between laboratories. This proposal primarily addresses the manual reading challenge by developing an instrument to record and analyze digital images of HA and HI assay plates. In Phase I, InDevR developed and launched a first generation instrument with an intuitive user interface and excellent HA and HI titer agreement with expert human readers. The Specific Aims for Phase II are to: 1) develop an advanced image analysis algorithm for challenging (seroepidemiology) samples, 2) enhance the Cypher HA/HI reader to create a walk-away high throughput system integrated with commercial automated plate handling equipment and sample tracking, and 3) validate reader performance at 3-5 facilities to demonstrate agreement with human experts and overall operational effectiveness. The advanced image analysis algorithm will analyze and compare specific features of control wells with sample wells. Preliminary results have shown greater than 95% agreement with expert human readers, including to ability to overcome non-specific inhibition and its effects on image interpretation. The new system will also support improved results transfer and LIMS integration. The high throughput system will be most valuable to laboratories like the FDA CBER`s group as well as WHO Collaborating Centers that select seasonal influenza vaccine antigens during peak periods using thousands of HA and HI assays.